MPK6‐mediated HY5 phosphorylation regulates light‐induced anthocyanin accumulation in apple fruit

Summary Light is known to regulate anthocyanin pigment biosynthesis in plants on several levels, but the significance of protein phosphorylation in light‐induced anthocyanin accumulation needs further investigation. In this study, we investigated the dynamics of the apple fruit phosphoproteome in response to light, using high‐performance liquid chromatography–tandem mass spectrometry analysis. Among the differentially phosphorylated proteins, the bZIP (basic leucine zipper) transcription factor, HY5, which has been identified as an anthocyanin regulator, was rapidly activated by light treatment of the fruit. We hypothesized that phosphorylated MdHY5 may play a role in light‐induced anthocyanin accumulation of apple fruit. Protein interaction and phosphorylation assays showed that mitogen‐activated protein kinase MdMPK6 directly interacted with, and activated, MdHY5 via phosphorylation under light conditions, thereby increasing its stability. Consistent with this finding, the suppression of the mitogen‐activated protein kinase genes MdMPK6 or MdHY5 resulted in an inhibition of anthocyanin accumulation, and further showed that light‐induced anthocyanin accumulation is dependent on MdMPK6 kinase activity, and is required for maximum MdHY5 activity. Under light conditions, active MdMPK6 phosphorylated MdHY5 leading to accumulation of phospho‐MdHY5, which enhanced the binding of MdHY5 to its target anthocyanin related genes in fruit. Our findings reveal an MdMPK6–MdHY5 phosphorylation pathway in light‐induced anthocyanin accumulation, providing new insights into the regulation of light‐induced anthocyanin biosynthesis in apple fruit at both the transcriptional and post‐translational levels.


Introduction
Anthocyanin pigments are a class of secondary metabolites and are responsible for the red, purple and blue colours of various organs in a wide range of plant species (Allan et al., 2008;Grotewold, 2006;Koes et al., 2005). Anthocyanins not only have multiple physiological functions in plants, such as aiding pollination and seed dispersal and adaption to adverse environmental conditions (Winkel-Shirley, 2001), but are also natural antioxidants with benefits as dietary components for human health, such as reducing cardiovascular disease and liver damage (Bondonno et al., 2019;Czank et al., 2013).
More than two decades of studies have revealed that anthocyanins are synthesized through the flavonoid pathway and that the associated biosynthetic genes are coordinately regulated by a MYB-bHLH-WD40/WDR (MBW) transcription factor (TF) regulatory complex (Albert et al., 2011;Ballester et al., 2010;Koes et al., 2005;Ramsay and Glover, 2005). The central role of the members of this complex in regulating anthocyanin biosynthesis has been documented in a broad range of land plant species (Allan et al., 2008;Koes et al., 2005;Ramsay and Glover, 2005). In apple (Malus domestica), three MYB TFs, namely, MdMYB1, MdMYB10, and MdMYBA, have been functionally characterized and shown to directly regulate the expression of anthocyanin biosynthetic genes (Ban et al., 2007;Espley et al., 2007;Takos et al., 2006). MdbHLH3, a lowtemperature-induced bHLH TF from apple, interacts with MdMYB1 and induces its expression and that of anthocyanin biosynthesis genes, thereby promoting anthocyanin accumulation (Xie et al., 2012). Recent studies showed that other TF families are also involved in anthocyanin synthesis, including members of the bZIP, NAC, Homo domain-leucine zipper (HD-ZIP), WRKY, and Ethylene-Response Factor (ERF) families, indicating a broad level of regulation at the transcriptional level (An et al., , 2018a(An et al., ,b, 2019aLiu et al., 2019;Sun et al., 2019).
In addition to temperature (Ubi et al., 2006;Xie et al., 2012), environmental factors such as light (Bai et al., 2019a,b;Fang et al., 2019) and nutrient availability (Jiang et al., 2007;Kadir and Sorkel, 2005;Schaberg et al., 2003;Sinilal et al., 2011), as well as exogenous hormones (An et al., 2018a,b;Wang et al., 2019), can influence anthocyanin biosynthesis by altering the transcription of anthocyanin-related genes. Light, an important signal for plant growth and development, has been particularly closely associated with inducing anthocyanin accumulation (Li et al., 2011). In apple, MdCOP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1), a specific E3 ubiquitin ligase for MYB1, enhances the expression of MdMYB1 to regulate anthocyanin biosynthesis under light conditions, and promotes the degradation of MdMYB1 via the ubiquitin pathway in the nucleus under dark conditions . The bZIP transcription factor HY5 (ELON-GATED HYPOCOTYL 5), acts as a central transcriptional regulator in light signalling by mediating photoreceptor responses to promote photomorphogenesis (Gangappa and Botto, 2016).
Recent studies also showed that HY5 is a positive regulator of anthocyanin accumulation through direct binding to the promoters of genes involved in anthocyanin biosynthesis. In tomato (Solanum lycopersicum L.), SlHY5 directly binds to the G-box and ACE motifs in the promoters of anthocyanin biosynthesis genes, such as CHS1, CHS2, and DFR and thereby regulates anthocyanin biosynthesis in fruit (Liu et al., 2018). In addition, MdHY5 functions upstream of light-induced MYB transcription and directly binds to the G-box motif of the MdMYB10 promoter to induce its expression, thereby also regulating light-induced anthocyanin biosynthesis . Finally, MdBBX20 encodes a B-box zinc finger protein that promotes anthocyanin accumulation synergistically with MdHY5 under light conditions . Despite all this knowledge, the mechanism of transcriptional HY5 regulation during light-induced anthocyanin accumulation is still not well understood.
In addition to transcriptional regulation, post-translational processes are critical factors for the output of gene expression. Protein phosphorylation is one of the most abundant and best understood post-translational modifications and affects signal transduction pathways by modulating protein activity and protein-protein interactions (Mithoe and Menke, 2011). Indeed, phosphorylation has been estimated to affect one-third of eukaryotic proteins (Qeli and Ahrens, 2010). Several studies have shown that protein phosphorylation is a part of light-induced developmental processes. For example, Arabidopsis thaliana MPK6 (mitogen-activated protein kinase 6) phosphorylates PIF3 (phytochrome-interacting factor 3) to regulate red light-induced cotyledon opening during seedling photomorphogenesis (Xin et al., 2018), and the A. thaliana MKK3-MPK6 cascade has been shown to be activated by blue light to regulate hypocotyl growth through phosphorylation of MYC2 (Sethi et al., 2014). Furthermore, our recent study showed that MdMPK4 can be activated in response to light, and that it phosphorylates MdMYB1 to increase its stability and promote anthocyanin accumulation (Yang et al., 2021), suggesting that MAPK-mediated protein phosphorylation contributes to anthocyanin accumulation.
A previous study showed that HY5 is also regulated by phosphorylation at its serine 36 residue, although the kinase responsible for this phosphorylation remains unknown . Furthermore, a recent study showed that SPA1 (Suppressor of phytochrome A-105) can directly phosphorylate HY5 and fine tune its stability and activity when regulating photomorphogenesis (Wang et al., 2021a). Taking these studies into consideration, we reasoned that HY5 phosphorylation may also be important for anthocyanin biosynthesis in apple fruit. In this study, we employed liquid chromatography (LC)-mass spectrometry (MS)/MS based label-free proteomics to show that HY5 was rapidly activated by light treatment of the fruit, and that MdMPK6 interacts with, and phosphorylates MdHY5. MdMPK6 functions to maintain the phosphorylation status of MdHY5, thereby preventing its ubiquitination by MdCOP1, leading to increased expression of anthocyanin related genes, elevated MdHY5 TF activity and increased anthocyanin accumulation.

Immunodetection of apple peel protein phosphorylation
In apple peels, light is considered to be a key factor in the regulation of anthocyanin synthesis (Espley et al., 2007). In our study, apples were bagged during growth and then transferred from the dark conditions to an incubator for light treatments. Apple peels showed visible signs of anthocyanin accumulation after 3 days of continuous light treatment (Figure 1a,b). To determine whether protein phosphorylation is involved in lightinduced apple pigmentation, apple (Malus domestica 'Red Fuji') fruit were treated with light for 0,5,10,15,30,40,60,120,240, and 360 min and then protein extracts from the corresponding peels were used for immunoblot analyses with phosphoserine and phosphothreonine antibodies. The intensity of the phosphorylation bands peaked in the 60 min sample, and then gradually decreased until the 360 min sample. Phosphorylated polypeptides of 23, 45, and 90 kDa were observed after 60 min light treatment, while only 23 and 45 kDa peptides were detected in response to light treatment for 360 min (Figure 1c).
To further confirm that the MAPK cascade is involved in fruit coloration, apple fruits were treated with a kinase inhibitor (K252a) or a phosphatase inhibitor (OA, okadaic acid). The OA treatment promoted anthocyanin accumulation and red coloration around the spraying site, whereas K252a treatment inhibited fruit coloration (Figure 1d). High-performance liquid chromatography (HPLC) analysis of anthocyanin contents further confirmed the phenotypic observation ( Figure 1e). Immunoblotting showed that K252a treatment caused a decrease in phosphorylation, while OA treatment caused a substantial increase in the signal of the phosphorylated bands, compared to the control (DMSO treatment; Figure 1f). These results indicated that light-induced anthocyanin accumulation in apple fruit is controlled by protein phosphorylation.
Label-free phosphorylation mass spectrometry identification of phosphorylated apple peel proteins following light treatment We profiled phosphoproteome changes in peels from apples that had been light treated for 0, 60 and 360 min. Phosphopeptides were enriched using TiO 2 affinity and identified in triplicate biological replicates by MS. A total of 3403 proteins and 7420 peptides were identified (Table S1), and 5003 phosphorylated peptides with 8634 phosphorylation sites, corresponding to 2901 phosphorylated proteins were recognized (Table S2). Analysis of the phosphorylated proteins revealed a significant difference in phosphorylation after light treatment. Furthermore, the number of phosphorylated proteins after a 60 min treatment was significantly higher than after a 360 min light treatment, indicating that the phosphorylation response occurred within a few hours. Using a log2(fold-change) > 1 and false discovery rate (FDR) < 0.05 cutoff, we identified 145 differentially phosphorylated proteins (DPPs) as being higher abundance up-regulated and 298 as being lower abundance in the 60 min sample compared with the 0 min sample. The total number of DPPs in 0 min vs. 360 min was 432, with 189 up-regulated and 243 down-regulated. For the 60 min vs. 360 min comparison, we found 279 DPPs, with 152 up-regulated, and 127 downregulated (Figure 2a), while there were 35 common DPPs between 0 min vs. 60 min, 0 min vs. 360 min and 60 min vs. 360 min, with 15 being up-regulated and 20 being downregulated ( Figure 2b). A total 798 DPPs were used to perform a PPI (protein-protein interaction) network analysis to investigate their relationships (Figure 2c). The confidence score was set at the highest level (≥0.900), and using the STRING database combined with Cytoscape, a complex PPI network was  (Table S3). Notably, the bZIP protein MdHY5 was found in the PPI network. MdHY5 has previously been shown to regulate light-induced anthocyanin biosynthesis in apple fruit, and to bind to the promoters of anthocyanin biosynthesis genes MdCHI and MdUFGT and the anthocyanin biosynthesis regulator MdMYB1 . We therefore targeted MdHY5 for further analysis.
To quantify the binding affinities of the MdMPK6 proteins to MdHY5, we compared the kinetic values of the interactions between MdMPK6-02G-MdHY5 and MdMPK6-15G-MdHY5 by biolayer interferometry (BLI). The KD value of the MdMPK6-02G-MdHY5 binding was higher than for the MdMPK6-15G-MdHY5 binding, suggesting that there is a relatively higher affinity between MdMPK6-15G-MdHY5 than between MdMPK6-02G-MdHY5. We deduced that MdMPK6-15G was mainly responsible for the interaction with MdHY5 in the fruit (Figure 3e).

Light-induced phosphorylation of MdHY5 requires MdMPK6
To test whether MdHY5 is an MdMPK6 substrate, an in vitro kinase assay was conducted. Proteins extracts were visualized using Coomassie Brilliant Blue (CBB) staining and the phosphorylated proteins were identified with an anti-phosphoserine/threonine antiserum ( Figure 4a). The results showed that MdHY5 is indeed phosphorylated by CA-MdMPK6-15G in vitro. Furthermore, in the presence of CA-MdMPK6-02G, 69HIS MBP-MdHY5 protein was not phosphorylated, suggesting that MdHY5 is not a MdMPK6-02G substrate. The MdHY5 phosphorylation site was next determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of TiO 2 chromatography-enriched MdHY5 phosphopeptides. Thirteen distinct phosphor-peptides were detected that corresponded to the MdHY5 amino acid sequence ( Figure 4b, Table S4). Previous studies showed that a single Ser-36 site was the main phosphorylation site of HY5, a site that was also found in MdHY5 phosphopeptides . To validate the Ser-36 site in vivo, it was mutated to Ala, and the phosphorylation level of MdHY5 and MdHY5 S36A recombinant proteins by CA-MdMPK6-15G were examined. The in vitro kinase assay also indicated that CA-MdMPK6-15G protein did not phosphorylate MdHY5 S36A (Figure 4c). To further confirm the MdHY5 phosphorylation site, we generated a phosphoantibody to the Ser-36 site. Immunoreactive bands were observed using immunoblot analysis of extracts from fruit exposed to light (0, 5, 10, 15, 30, 40, and 60 min; Figure 4d), and the amino acid sequences of the immunoreactive bands were shown by LC-MS/MS to include the Ser-36 site. MdHY5 phosphorylation increased after a 5 min light treatment, and the highest abundance of the phosphoprotein was observed after 10 min of light treatment, and the expression level of MdHY5 was continuous increased with light treatment ( Figure S4), which further suggested that MdHY5 is rapidly activated by light treatment (Figure 4d).

MdMPK6 proteins participate in the accumulation of anthocyanin in apple peels
To demonstrate that MdMPK6 is involved in light induced anthocyanin accumulation in apple peel, we used the pGUS-PLUSPLUS vector to construct OE-MdMPK6-15G, OE-CA-MdMPK6-15G, and used the TRV2 silencing vector to construct TRV2-MdMPK6. Agrobacterium infiltration was used to transfer and integrate the vectors into apple peels. As shown in Figure 5a, OE-MdMPK6-15G and OE-CA-MdMPK6-15G caused anthocyanin enrichment, and there was no significant anthocyanin accumulation in TRV2-MdMPK6-infiltrated apple fruit. HPLC results supported the phenotypes in infected apple fruit ( Figure 5b). We also observed that the expression of anthocyanin biosynthesis genes MdCHI and MdUFGT and the anthocyanin biosynthesis regulator MdMYB1 was decreased in MdMPK6silenced fruit than in the control, and MdCHI and MdUFGT was induced in MdMPK6-overexpressing apple fruit than in the control, partially confirming them to be MdHY5 targets ( Figure 5c, Figure S5).
The above results demonstrate that MdMPK6 can interact with, and phosphorylate MdHY5 and so we hypothesized that the increase in anthocyanin levels in OE-MdMPK6 was accompanied by phosphorylation of MdHY5. To test this, we analysed the phosphorylation level of MdHY5 in the infiltrated apple fruit. Lower MdHY5 protein phosphorylation levels were detected in  To explore whether MdMPK6 promoted light-induced anthocyanin synthesis depends on the phosphorylation level of MdHY5, the sequences of MdHY5 (native form), a phosphomimic MdHY5 S36D form (Ser-36 mutated to Asp), and a phosphodeficient MdHY5 S36A form (Ser-36 mutated to Ala) were inserted into the pGFPGUSPLUS vector downstream from the 35S promoter. Phenotypic analysis showed that high levels of expressions of MdHY5 S36D and MdHY5 in apple fruit promoted anthocyanin accumulation and red coloration around the injection sites when compared to the control infiltrated fruit. In contrast, expressing OE-MdHY5 S36A did not raise obviously anthocyanin accumulation (Figure 5a,b). When we examined the RNA abundance of the three MdHY5 target genes MdMYB1, MdCHI and MdUFGT, the highest levels were observed in OE-MdHY5 S36D infiltrated fruit (Figure 5c). Immunoblot analysis showed that a strong and a weaker band, respectively, of phosphorylated MdHY5 were detected in MdHY5 S36D and MdHY5 S36A infiltrated fruit ( Figure 5d). These results suggested that MdMPK6 participates in anthocyanin biosynthesis by phosphorylating MdHY5, and that Ser-36 is the predominantly phosphorylated residue.
Subsequently, anti-MdHY5 and anti-phosphorylated-MdHY5 antibodies were used for immunoblotting. The result showed that OE-MdMPK6-15G, OE-CA-MdMPK6-15G, OE-MdHY5, and OE-MdHY5 S36D transgenic apple calli exhibited stronger MdHY5 phosphorylation than the control and other transgenic calli. At the same time, weaker immunoreactive bands were observed in wild type, OE-MdHY5 S36A and RNAi-MdMPK6 transformed calli (Figure 6d), so we deduced that MdHY5 contributes to lightinduced-promoted anthocyanin biosynthesis in an MdMPK6dependent manner.

MdMPK6 prevent MdHY5 degradation under dark conditions
Next, we examined the phosphorylation level of MdHY5 in K252a-and OA-treated apple calli. We observed that phosphorylated MdHY5 protein levels were substantially reduced after K252a treatment compared with the control (DMSO treatment), while they were significantly increased after OA treatment ( Figure 7a). These results suggested that phosphorylation of MdHY5 is mediated by the MAPK cascade.
Previous studies showed that light-mediated HY5 phosphorylation provides an added level of HY5 stability, and that unphosphorylated HY5 shows stronger interaction with COP1 in darkness, where it is also degraded via the proteasome pathway by the COP1 protein Wang et al., 2021a). We evaluated the effect of MdMPK6-mediated MdHY5 phosphorylation on MdHY5 stability using OE-MdHY5, OE-MdHY5 S36A , and OE-MdHY5 S36D transgenic apple calli treated with MG132, a 26S proteasome inhibitor under dark conditions. The results showed that MdHY5 S36D accumulated to high levels than that in MdHY5 and MdHY5 S36A in dark treated apple calli (Figure 7b). Our results indicated that MdMPK6-mediated MdHY5 phosphorylation affects protein stability under dark conditions. Finally, we performed in vitro pull-down assays to examine whether the phosphorylation of HY5 affects its interactions with MdCOP1. We observed that the MdHY5 S36A protein had a higher affinity for MdCOP1, whereas the MdHY5 S36D protein has a lower affinity to MdCOP1 than to MdHY5 (Figure 7c). Taken together, our results showed that phosphorylation alters the affinity of HY5 for COP1 and affects its accumulation.

MdMPK6-15G phosphorylate MdHY5 in the fruit ripening stage under light conditions
To further understand the biological function of MdMPK6-15G phosphorylation of MdHY5 in apple fruit, we measured MdHY5 accumulation during fruit ripening. In 'Royal Gala' fruit, coloration was present in the fruit-ripening stage (100 DAFB, days after full bloom), and HPLC results confirmed the presence of anthocyanins (Figure 8a,b), so we focused on the 60, 80, and 100 DAFB stages for subsequent analysis.
To characterize MdHY5 expression, we analysed the MdHY5 promoter region up to 2000 bp upstream from the translation  in infiltrated apple fruit between 9 am and 9 pm from 60 to 100 DAFB. Actin was used as a protein loading control. RT-qPCR, high-pressure liquid chromatography (HPLC) and immunoblot analysis were performed using three biological replicates. Data are means AE SD of three independent biological replicates. Different letters above the bars indicate significantly different values (P < 0.05), calculated using one-way analysis of variance (ANOVA) followed by a Tukey's multiple range test. start site using the PlantCARE database. Several light-responsive elements were identified in the MdHY5 promoter (including a chs-Unit 1 m1; I-box; G-box), and we also identified a TGACGmotif involved in methyl jasmonate (MeJA) responses, which suggested that MdHY5 may participate in fruit ripening ( Figure 8c). Next, we determined MdHY5 transcript levels in 3-h intervals over a 24-h period at 60, 80, and 100 DAFB. MdHY5 transcript levels exhibited a diurnal expression pattern, with higher expression during the day and lower expression at night, reaching a peak at 3 or 6 pm (Figure 8d).
To determine whether MdMPK6-15G-phosphorylated MdHY5 protein was also present in diurnal pattern, we assessed the abundance of MdHY5 and the phosphorylated form in the samples collected at 9 am and 9 pm at the different developmental stages. Immunoblot analysis indicated a large increase in the accumulation of MdHY5 in the fruit ripening stage (100 DAFB), and a higher abundance at 9 am than at 9 pm. This corresponded to the pattern of MdHY5 transcript levels ( Figure 8d). MdHY5 phosphorylation levels in 60 and 80 DAFB were higher at 9 pm than at 9 am, but higher abundance at 9 am than at 9 pm was observed in the fruit ripening stage (100 DAFB), as examined using anti-Phos-MdHY5 antibodies. Meanwhile, MdHY5 phosphorylation levels correspond to the MdHY5 protein abundance (Figure 8e). To summarize, these results indicated that MdMPK6-15G phosphorylation of MdHY5 mainly occurred in the fruit ripening stage and that this promoted anthocyanin accumulation in the daytime.

MdHY5 functions synergistically with MdMPK6 to modulate anthocyanin accumulation in apple fruit
To further test whether MdMPK6-15G and MdHY5 work together to regulate anthocyanin biosynthesis during fruit coloration, we transiently overexpressed or silenced MdMPK6-15G in the corresponding MdHY5 transient expressed fruit. Elevated expression of MdMPK6-15G and CA-MdMPK6-15G in MdHY5-silenced fruit had no significant effect on anthocyanin accumulation, while overexpressing MdMPK6-15G and CA-MdMPK6-15G in OE-MdHY5 and OE-MdHY5 S36A fruit significantly promoted anthocyanin accumulation compared to MdHY5s-overexpressing fruit. When CA-MdMPK6-15G was overexpressed in OE-MdHY5 S36D fruit, the highest anthocyanin accumulation was observed. However, silencing of MdMPK6-15G and CA-MdMPK6-15G significantly suppressed anthocyanin accumulation in MdHY5, MdHY5 S36A , and MdHY5 S36D over-expressing fruit (Figure 9a,b). RT-qPCR analysis showed that overexpressing MdMPK6-15G and CA-MdMPK6-15G led to higher expression of biosynthesis and regulatory genes involved in anthocyanin accumulation in OE-MdHY5, OE-MdHY5 S36A , and OE-MdHY5 S36D fruit. MdMPK6-15G and CA-MdMPK6-15G silencing in MdHY5, MdHY5 S36A , and MdHY5 S36D over-expressing fruit also suppressed the expression of anthocyanin biosynthesis and regulatory genes, although to a lesser degree (Figure 9c). These results indicated that the function of MdHY5 in regulating anthocyanin biosynthesis is dependent on its phosphorylation by MdMPK6.
Furthermore, to explore the MdMPK6 effect on MdHY5 function to regulate anthocyanin-related gene expression, dualluciferase (LUC) analysis was used to detect the expression of LUC driven by the MdMYB1, MdCHI, and MdUFGT promoters. Different MdHY5 and MdMPK6-15G effector constructs were expressed under control of the 35S promoter, and cotransformed with proMdCHI, proMdUFGT or proMdMYB1:: GUS reporter constructs and the internal control (35S::LUC) into N. benthamiana leaves (Figure 10). We observed that MdHY5 activated the expression of MdMYB1, MdCHI, and MdUFGT, and LUC activities for the MdMYB1, MdCHI, and MdUFGT promoters were significantly greater when MdMPK6-15G and MdHY5 were co-expressed. MdHY5 and MdHY5 S36D displayed similar transcriptional activation ability, whereas MdHY5 S36A did not cause enhanced transcriptional activation of the anthocyanin related gene promoters.

Discussion
Fruit coloration is controlled by the concerted action of various signalling pathways that integrate information regarding environmental conditions with that from developmental and metabolic cues (Field et al., 2001). Among these signals, light is a key factor in triggering anthocyanin biosynthesis (Kami et al., 2010). Physiological and biochemical studies have demonstrated that lightinduced fruit coloration is regulated by several MYB TFs in apple fruit. The apple MdMYB10/MdMYB1/MdMYBA TFs are thought to be activated by light and promote anthocyanin accumulation by activating the transcription of anthocyanin biosynthesis genes (Ban et al., 2007;Espley et al., 2007;Takos et al., 2006). Although a light-induced anthocyanin biosynthesis mechanism has long been suggested, the identification of upstream regulatory components has been limited.
Plants have evolved four classes of photoreceptors to monitor the surrounding light conditions: red/far-red light-sensing phytochromes, blue/ultraviolet (UV) A light-sensing cryptochromes and phototropins, and UVB light-sensing UVR8 (Chen et al., 2004;Paik & Huq, 2019). Notably, all the light signals perceived by different photoreceptors converge on the central downstream transcription factor elongated hypocotyl 5 (HY5). In A. thaliana, more than 60% of early-induced genes by phyA or phyB are HY5 direct targets, which is consistent with the notion that HY5 is one of the highest ranking regulators in the hierarchy of the transcriptional cascade that control diverse programs such as photomorphogenesis, nutrient signalling, and abiotic and biotic stress responses (Gangappa and Botto, 2016). Several studies have reported that HY5 is involved in the regulation of anthocyanin biosynthesis Jiang et al., 2016;Shin et al., 2013). In tomato, SlHY5 regulates light-induced anthocyanin accumulation by directly binding to the promoters of anthocyanin biosynthesis genes, such as CHS1, CHS2, and DFR (Wang et al., 2021b). In addition, loss of function of tomato HY5 (SlHY5) was reported to impair pigment accumulation, and a chromatin immunoprecipitation assay showed that anthocyanin biosynthesis genes are direct targets of SlHY5 (Wang et al., 2021a). In apple fruit, MdHY5 induces anthocyanin accumulation and positively regulates both its own transcription and that of MdMYB10 by binding to E-box and G-box motifs, respectively . We, therefore, deduced that HY5 is functionally located upstream from MdMYB10 and is a hub TF in anthocyanin biosynthesis transcriptional networks. Here, we confirmed that MdHY5 promoted light-induced anthocyanin biosynthesis by overexpressing it into apple calli and fruit. Furthermore, yeast one-hybrid (Y1H) assays and transient expression results showed that MdHY5 can bind to the promoters of the anthocyanin related genes MdCHI, MdUFGT, MdMYB10, and activate their transcription. Together, these results suggest that MdHY5 is located upstream of the anthocyanin biosynthesis transcription regulatory pathway and mediates light induced anthocyanin accumulation in apple fruit.   In a previous study, HY5 was found to be regulated by phosphorylation of the serine 36 residue . The unphosphorylated form of HY5 interacted more strongly with COP1 and was preferentially degraded under dark conditions, while it became more abundant in light-grown seedlings , which suggested that phosphorylation may be essential for the activity of HY5 during light induced developmental processes. Recently, a study further confirmed that SPA proteins can directly phosphorylate HY5 and fine-tune its stability and activity in regulating photomorphogenesis (Wang et al., 2021a). We hypothesized that a similar regulatory mechanism might be present in apple fruit. Our results showed that a light-induced protein kinase, MdMPK6, phosphorylates MdHY5 under light treatment to promote its transcriptional activity in apple fruit. MdMPK6 overexpression in apple fruit and calli revealed that MdMPK6 enhances anthocyanin accumulation and induces MdHY5 protein activity in response to light. Previous results showed that Ser-36 is the phosphorylation site in A. thaliana , and the same was true for MdMPK6 phosphorylation of MdHY5 in apple. Phosphor-inactive and mimic analyses indicate that MdHY5 activity in light-induced anthocyanin accumulation is enhanced when MdHY5 is mutated to MdHY5 S36D , and abolished when MdHY5 is mutated to MdHY5 S36A . We conclude that MdMPK6-mediated MdHY5 phosphorylation of Ser-36 induces anthocyanin accumulation in fruit. We also noticed that MdMPK6 overexpression in apple promote the transcription level of MdHY5. Previously studies showed that HY5 has also been reported to feed back to promote its own transcription (Abbas et al., 2014;Binkert et al., 2014;Burko et al., 2020). So, we deduced that massive expression of MdMPK6 promoted the protein activity of MdHY5, and MdHY5 promote its own expression.
Phosphorylation or ubiquitination of TFs provides a versatile means to regulate protein activity during plant development, and can enhance or suppress biological processes (Hunter, 2007). For example, in apple, the MdbHLH3 TF is phosphorylated by the protein kinase MdHXK1 in response to glucose signals, and ubiquitinated by high-glucose-inhibited U-box-type E3 ubiquitin ligase MdPUB29, to regulate ethylene biosynthesis and hence fruit quality (Hu et al., 2016. The HY5 ubiquitination mechanism has been well studied during plant photomorphogenesis. Here, HY5 stability is directly proportional to increased light intensity and, in darkness, its turnover is mainly regulated by the CONSTITUTIVE PHOTOMORPHOGENIC 1 (COPI)-mediated 26S proteasome pathway . A recent study also showed that HY5 phosphorylation results in lower binding affinity to COP1 and SPA1, which reduces HY5 ubiquitination and, in turn, stabilizes HY5 proteins in the dark (Wang et al., 2021a). Here, a similar regulatory mechanism involving MdHY5 was uncovered in apple fruit. MdHY5, as a major light responsive regulator, can be ubiquitinated by MdCOP1 and phosphorylated by MdMPK6 to coordinate its own protein abundance, leading to the regulation of anthocyanin. Our previous study found that phosphorylation of the anthocyanin biosynthesis regulator MdMYB1 by MdMPK4 inhibits the MdCOP1-MdMYB1 interaction and prevents ubiquitination of MdMYB1 under light conditions (Yang et al., 2021). Thus, phosphorylation or ubiquitination provide an adaptable mechanism in light-mediated signalling and facilitate the accumulation of anthocyanin biosynthesis regulators, thereby enhancing anthocyanin accumulation when fruit are exposed to light.
In a previous study, we found two MdMPK4 genes in the apple genome, indicating that the MAPK gene family has expanded in apple (Yang et al., 2021). MdMPK4-06G was more responsive to light treatment and catalysed phosphorylation of MdMYB1 in fruit, thereby modulating anthocyanin biosynthesis (Yang et al., 2021). MdMPK4-14G can be activated by exposure to darkness and is involved in the dark-induced degreening of fruit peels via MdERF17 phosphorylation (Wang et al., 2022). MdMPK4 proteins thus act as molecular bridges in both chlorophyll degradation and anthocyanin accumulation during apple fruit ripening. Two MdMPK6 proteins were also identified in the apple genome, and the binding ability of MdMPK6-15G to MdHY5 was found to be higher than that of MdMPK6-02G to MdHY5. Moreover, only MdMPK6-15G was found to phosphorylate MdHY5, so we hypothesized that, as with MdMPK4 proteins, the two MdMPK6 proteins have different functions, with MdMPK6-02G participating in photomorphogenesis by phosphorylating other proteins. Future studies will elucidate the functions of the two MdMPK6 copies.
In summary, our data reveal MdHY5-mediated regulatory mechanism for promoting light induced anthocyanin accumulation, wherein MdMPK6 phosphorylates MdHY5 (Figure 11). During exposure of apple fruit to light, MdMPK6-15G phosphorylates MdHY5 at Ser-36 to activate its transcriptional activity. Phosphorylation of MdHY5 prevents it degradation by MdCOP1 and so increases its stability. These results suggest potential strategies for the biotechnological fine-tuning of anthocyanin accumulation during light-induced anthocyanin accumulation, and for guiding traditional breeding programs in creating new cultivars with improved fruit coloration.

Plant materials
Bagged apple (Malus domestica cv. 'Red Fuji') fruit were harvested 160 days after full bloom (DAFB) from the Germplasm Resource Nursery of Malus in Shunyi District, Beijing University of Agriculture (116°E, 40°N). The bagged fruits were placed in a dark incubator for 24 h at 23°C to equilibrate the conditions. For light treatment, the harvested apples were transferred under low light from their bags to a white light incubator (390-780 nm, 215 lmol/m 2 /s 1 , 23°C). Fruit peels were collected and frozen in liquid nitrogen for later analysis, which was conducted in three biological replicates. Apple calli were induced from young embryos of the 'Orin' apple cultivar (M. domestica Borkh.). Calli were sub-cultured in MS medium containing 0.5 mg/L indole-3acetic acid (IAA) and 1.5 mg/L 6-benzylaminopurine (6-BA) at 25°C in the dark. The apple calli were sub-cultured three times at 15-day intervals before being used for genetic transformation and other assays . Light treatment of debagged apple fruit (160 DAFB, before full ripeness, immediately after 1 day of dark treatment) and apple calli were performed by placing the samples under white (390 nm-780 nm, 12 000 lux) light. Nicotiana benthamiana was grown in a greenhouse at 25°C under a 16 h day, 8 h night cycle.
Protein extraction, peptide digestion, and peptide fractionation Proteins were extracted from the apple fruit peels (three biological replicates per treatment) and homogenized in liquid nitrogen. Specifically, 20 g of apple peel sample, corresponding to approximately 2 mg protein, were added to a lysis buffer (4% SDS, 100 mM Tris/HCl pH 7.6, 0.1 M dithiothreitol (DTT), 5 mM sodium fluoride, 50 mM b-glycerophosphate, 1 mM sodium orthovanadate, supplemented with the HALT protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, San Jose, CA, 78441)). Two milligrams of protein extract were digested with endoproteinase Lys-C followed by trypsin according to the MED-FASP method (Wi sniewski and Mann, 2012). The trypsin digestion was stopped by adding trifluoroacetic acid (TFA) to a final concentration of 1% (vol/vol) and placed on ice then reconstituted in a 2% acetonitrile (ACN), pH 10 (pH adjusted using an ammonia solution (Wako, 013-23355)) and centrifuged at 10 000 g to clarify the mixture. The peptides were then separated by basic-pH reverse-phase liquid chromatography (basic pH RPLC) using an Agela Durasbell C18 column (150°A, 5 lm, 4.6 9 250 mm) and a RIGOL L-3000 HPLC. A gradient of solvent A (2% ACN, pH 10) and solvent B (98% ACN, pH 10) was used to separate the peptides based on their hydrophobicity at pH 10, as in Ren et al. (2018).
Enriched phosphopeptides were resuspended in 10 lL of 10% FA and subjected to online reverse phase nano LC-MS/MS analysis with 50% of sample loading using an Easy nLC 1000 (Thermo Fisher Scientific), coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific; Ren et al., 2018).

Bioinformatics analysis
Raw files were processed with MaxQuant (version 1.3.0.5) and aligned to the apple (Malus domestica) reference genome (Velasco et al., 2010). Protein function was annotated using the Blast2GO software (http://www.blast2go.com/b2ghome). The phosphopeptides were centered around the phosphorylated amino acid residues and aligned with seven positions upstream and downstream from the phosphorylation sites included. All differentially phosphorylated proteins that were classified as identifiers were queried for PPIs in the STRING database version 10.5 (http://string-db.org/cgi/input.pl).
Cloning of the MdMPK6 genes and RT-qPCR analysis 'Red Fuji' apple peel cDNA was used as a template to clone the full-length MdMPK6 and MdHY5 genes. Genomic DNA was isolated from fruit peel to analyse MdHY5 promoter sequence. Cis-elements were predicted using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and the primers are listed in Table S5. Sequence comparisons and analyses were conducted as previously described (Kumar et al., 2004).
Gene expression levels were analysed by RT-qPCR using the Premix Ex Taq TM kit (Probe qPCR) (TaKaRa, Ohtsu, Japan), and a Bio-Rad CFX96 Real-Time PCR System (BIO-RAD, Hercules, CA), according to the manufacturer's instructions. MdActin (LOC103453508) and AtActin (AT3G46520) were used as the internal controls. The data were analysed using the internal controls and the 2 (ÀΔΔCt) method (Livak and Schmittgen, 2001). The threshold cycle (Ct) value of MdActin was subtracted from that of the gene of interest to obtain a DCt value. The Ct value of control sample was subtracted from the DCt value to obtain a DDCt value. The fold changes in expression level relative to the control were expressed as 2 (ÀΔΔCt) (Livak and Schmittgen, 2001). The mean AE SD is calculated by taking the mean of three biological replicates and then taking the mean of the individual samples. Analysis of the fruit, leaves and calli samples was repeated with three biological replicates. All the primer and probe primer sequences are listed in Table S5.

Anthocyanin quantification
The HPLC analysis was used to determine apple peel anthocyanin content. The extraction was conducted as previously described (Tian et al., 2017), and anthocyanins were quantified by measuring absorbance at 520 nm using a calibration curve based on a commercial cyanidin-3-O-glucoside standard (Sigma, St Louis, MO). The anthocyanin concentration was expressed as lg/ g of fresh weight (FW; Revilla and Ryan, 2000). All the samples were analysed using three biological replicates (extracted from the peels of three different apples, calli, leaves, or plants).

Y2H, BiFC, pull-down, Co-IP and BLI assays
For directed Y2H assays, the MdHY5 coding sequence was cloned into the pGADT7 vector as an activation domain, and MdMPK3s, MdMPK4s, MdMPK6s; and the constitutively active forms CA-MdMPK3s, CA-MdMPK4s, and CA-MdMPK6s were cloned into the pGBKT7 vector as baits. Assays were performed according to the manufacturer's instructions (Invitrogen, Carlsbad, CA).
MdHY5-GST, MdHY5-69HIS, CA-MdMPK6-69HIS, and MdMPK6-GST proteins were expressed in Escherichia coli for use in a GST pull-down assay. Co-IP was performed as previously described (Fiil et al., 2008). MdHY5, MdMPK6, and CA-MdMPK6 were generated as C-terminal fusion proteins with HA or GFP tags. The elution products were analysed by Western blot analysis . BLI assays were conducted on an Octet RED 96 System, according to the manufacturer's protocol (Gu et al., 2016).

Recombinant protein purification
To promote the solubility of MdHY5, the coding sequence was inserted into the pGEX4T-2, pETM-41 vector (TaKaRa, Ohtsu, Japan; Qing et al., 2004) for low-temperature induction. pGEX4T-2-MdHY5 and pETM-41-MdHY5 were transformed into E. coli (Rosetta strain) for in vitro induction with 0.5 mM IPTG (isopropylthio-b-galactoside) overnight at 16°C. The MdHY5 protein was purified on a GST-Trap, HIS-Trap column and an AKTA purifier automatic chromatograph (GE Healthcare, Bucks, UK). The MdMPK6 coding sequence was introduced into the pGEX4T-2 and pETM-41 vectors and transformed into E. coli (Rosetta), and the bacterial cultures were induced with 0.5 mM IPTG overnight at 16°C. The MdMPK6 protein was purified on GST-Trap and HIS-Trap columns with an AKTA purifier automatic chromatograph (GE Healthcare).

Phosphorylated antibody preparation
The antibodies for anti-phosphorylated-MdHY5 and anti-MdHY5 were prepared by Beijing QiWei YiCheng Tech Co., Ltd. First, phosphorylated peptides and nonphosphorylated peptides of DEMESSEGMESDEEIGR were synthesized to a purity of over 90%. The phosphorylated peptides and non-phosphorylated peptides were coupled with KLH to immunize rabbits. After the sixth immunization, an antiserum sample was taken to test the serum titre, and after the eighth immunization, the antiserum samples were collected. Second, a nonphosphorylated antigen affinity purification column was used to purify the antiserum to obtain antibodies recognizing the nonphosphorylated peptides until the peak of antibodies reached a certain low level. After all these antibodies were removed, a phosphorylated antigen affinity purification column was used to purify the antiserum to obtain the antibody recognizing the phosphorylated peptides.

In vitro phosphorylation assays and LC-MS/MS analysis
In vitro phosphorylation assays were performed as previously described (Andreasson et al., 2005). For MS analysis, 2 lg of recombinant MdHY5 protein was mixed with 1 mg of recombinant CA-MdMPK6-02G or CA-MdMPK6-15G protein in kinase reaction buffer containing 1 mM ATP. Reaction conditions and related analysis methods were as previously described . Phos-tag analysis was performed as previously described (Yin et al., 2018).

Construction of expression vectors and stable transformation of apple calli
Overexpression vectors were made by amplifying the full-length MdMPK6-15G and MdHY5 sequences from 'Red Fuji' apple peel cDNA, using RT-PCR. CA-MdMPK6-15G, MdHY5 S36A and MdHY5 S36D were generated using a site-directed mutagenesis kit (TaKaRa, Ohtsu, Japan) and confirmed by sequencing. The corresponding cDNA sequences (MdMPK6-15G, CA-MdMPK6-15G, MdHY5 S36A , MdHY5, and MdHY5 S36D ) were cloned into the pGFPGUSPLUS plant transformation vector (Vickers et al., 2007) under the control of the CaMV 35S promoter. The RNAi vector (pK7GWIWG2) was made by amplifying the MdMPK6-15G (1221 bp) and MdHY5 (518 bp) DNA fragments from 'Red Fuji' apple peel cDNA (Zhao et al., 2019). The DNA fragments were cloned into the pDONR207 entry vector and then assembled into the pK7GWIWG2 destination vector using Gateway recombination cloning technology (Thermo Fisher Scientific, Wilmington, DE). The constructed vector was transformed into Agrobacterium (LBA4404), which was then used to infect the calli to obtain the silenced tissue. All primers are listed in Table S5. Transformation of 'Orin' calli was performed as previously described . Three independent transgenic apple calli lines were obtained.

Transient expression in apple fruit and tobacco leaves
Bagged 'Red Fuji' apple fruit were harvested 140 days after bloom for transient expression. TRV vectors and Agrobacterium were constructed as previously described . The infiltration protocol and culturing methods for transient expression assays were adapted from previously described methods (Liu et al., 2002;Zhang et al., 2016). The infected apples ('Red Fuji') were placed at 23°C for either 2 days (for overexpression) or 5 days (for silencing). Infiltration was repeated using three biological replicates.
Dual-luciferase assays were used to analyse trans-activation of target gene promoters by TFs as previously described (Hellens et al., 2005;Xiang et al., 2015). The full-length MdHY5 sequence and the MdMYB1, MdCHI, and MdUFGT promoters were cloned into the pGreenII-62-SK and pGreenII-0800-LUC vectors, respectively, with the primers listed in Table S5. The recombinant plasmids were transformed into Agrobacterium strain GV3101. Cultures expressing TFs and promoters were mixed (10:1 v/v) and then infiltrated into tobacco (N. benthamiana) leaves (Hellens et al., 2005). Firefly luciferase and Renilla luciferase enzyme activities were tested using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). The enzyme activity assay was carried out with three biological replicates.
Treatment of apple calli with kinase inhibitor (K252a) and phosphatase inhibitor (OA) To determine the effects of K252a (kinase inhibitor) and OA (phosphatase inhibitor) on MdMPK6-MdHY5 phosphorylation, calli expressing MdMPK-15G and MdHY5 were light-treated and phosphorylation levels of the two proteins were determined. Stably transformed calli were transferred to MS medium supplemented with 10 lM K252a or 1 lM OA (K252a #12754; Okadaic Acid #5934, Cell Signalling Technology, Danvers, MA), and cultured for 3 days in the dark. Wild-type calli were used for comparison. The calli were treated with strong light and proteins were extracted as described above for Western blot analysis. Apple fruit were sprayed with 10 lM K252a or 1 lM OA and then stored in a growth room at 22°C under natural light conditions with a daylight period of 16 h for 3 days. Three biological replicates were analysed.

Treatment of apple calli with MG132
Apple calli were placed under high light for 2 days, prior to MG132 treatment. Apple calli were placed in medium with MG132 at a concentration of 100 lM and an aqueous solution of DMSO was used as the control treatment . The treated apple calli were placed in the dark for 12 h, and then proteins were extracted as described above for Western blot analysis. The experiments were carried out with three biological replicates.

Data analysis
All the data were analysed using one-way ANOVA followed by Tukey's multiple range test to compare differences between the experimental data at P < 0.05. Origin95, Microsoft Excel 2016 and IBM SPSS Statistics 22 were used for analyses. Quantification of band intensity was performed using ImageJ (https://imagej.nih. gov/ij/).

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article.

Figure S1
Alignment of MPK3, MPK4, and MPK6 amino acid sequences in different species. Asterisks represent identical amino acids. Figure S2 Y2H analysis of the interaction between MdMPK3 and MdMPK4 proteins and MdHY5. Figure S3 Negative control of BiFC assay. Co-transformation of MdMPK3s and MdMPK4s with MdHY5 proteins into tobacco cells, respectively. Figure S4 Expression analysis of MdHY5 under light treatment. RT-qPCR was performed using three biological replicates. Data are means AE SD of three independent biological replicates. Different letters above the bars indicate significantly different values (P < 0.05), calculated using one-way analysis of variance (ANOVA) followed by the Tukey's multiple range test.